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Detection of Antibiotics Residues in Raw milk

Produced in Freha Area (Tizi-Ouzou), Algeria

Article January 2013

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Bulletin UASVM, Veterinary Medicine, 70(1)/2013
Print ISSN 1843-5270; Electronic ISSN 1843-5378

Detection of Antibiotics Residues in Raw milk Produced in Freha Area

(Tizi-Ouzou), Algeria.

TITOUCHE Y1., A. HAKEM (EX AKAM)1 , K. HOUALI 3, B. YABRIR1,

O. MALKI2, A. CHERGUI3, N. CHENOUF1, S. YAHIAOUI2, M. LABIAD1, H. GHENIM2,
S. KECHIH-BOUNAR2, F. CHIRIL 4, G. NAD 4 and N.I. FI4.
1
Laboratoire dExploration et Valorisation des Ecosystmes Steppiques,Universit Djelfa, Algeria.
2
Laboratoire Rgional Vtrinaire, Draa-Ben-Khedda, Tizi-Ouzou, Algeria,
3
Laboratoire de Biotechnologie et de Biochimie Analytique,
Universit Mouloud Mammeri, Tizi-Ouzou, Algeria.
4
University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Romania
Corresponding author: yacinetitouche@yahoo.fr

Abstract. The misuses and uncontrolled veterinary drugs in animals food production can have
harmful effects on consumers health and dairy industry. The presence of antibiotic residues in milk
can cause partial or total inhibition of the growth of lactic acid bacteria, which can induce
technological flaws on the cheese production. The aim of this study is to detect antibiotic residues in
raw milk produced in Freha area (Tizi-Ouzou) in Algeria. A total of 171 milk samples were
collected from 14 dairy farms and examined. The first screening of the samples was carried out by
acidification test using Bacillus stearothermophillus (variety calidolactis ATCC 10149), followed
by a confirmation test agar with spores of B. stearothermophillus, B. subtilis and B. megaterium.
Our results showed a strong presence of antibiotic residues in raw milk, with 80 positive samples
(46.78%). Most of them contained penicillin and/or tetracycline (88.75%), followed by macrolide
and/or aminoglycoside (12.5%). In contrast, the sulfonamides were only present in 5% of the
positives cases. The results indicate that most of the farmers do not always respect the time delay
between the administration of antibiotics and the milk collection. Therefore, the control of antibiotic
residues must be a major concern for producers and processors to protect healths consumers.
Keywords: antibiotics residues, raw milk, microbiology, acidification test, confirmation test.

INTRODUCTION

Applied to milk, the term quality includes a range of concepts. It involves the
proportions of main constituents, its chemical composition and organoleptic properties, the
presence or absence of pathogenic germs, as well as exogenous substances, which can also
affect the healths consumers (Ekman, 2000). Modern breeding farms use a wide range of
veterinary drugs. These antibiotics are used either as growth promoters or as therapeutic
remedies. Overuse and non respect of time between their administration in animal and the
milk collection, may induce fortuitously milk contamination. Their presence in human foods
is associated with several adverse public health effects, including hypersensitivity,
gastrointestinal disturbance and neurological disorder. Another aspect of the problem is the
fact that the presence of antibiotics in milk can prevent fermentation involved in the
production of some basic foodstuffs such as yogurt and cheese (Rogister et al., 2002).
Several methods have been described in the literature for the detection of antimicrobial
substances in milk. Those differ in their sensitivity, their implementation and their cost. The
control of antibiotic residues in milk can be checked after using a qualitative method such as
microbiological screening methods and immunological tests but also with using a quantitative
test as the analytical methods (liquid chromatography coupled with UV spectroscopy or mass

83
fluorimetry) (Pericas et al., 2010). The purpose of this study is to determine the incidence of
antibiotic residues in raw milk produced in our area in order to have some information about the
abusive using of veterinary drug in dairy cattle and to assess the risk for the healths consumers.

MATERIALS AND METHODS

Study area and sampling conditions: This study involved a total of 171 individual raw
milk samples collected from 14 dairy farms in Freha (Tizi-Ouzou area) in Algeria. All
samples came from declared healthy cows by the farmer and not undergone any antibiotic
treatment three weeks before. This milk is intended for human consumption or is delivered to
different dairies. The raw milk samples were collected in sterile plastic bottles and sent to the
laboratory in the same day, using refrigerated cooler and stacked ice-bag, stored at 4 C.
Before being tested, all raw milk samples were heat treated at 80 C for 10 minutes in order to
inactivate all inhibitory substances naturally present in the milk.
Treatment of samples: The detection of antibiotic residues in samples was performed
according to the official European method for detecting antibiotic residues in milk
(Commission Decision 91/180/EEC of 14 February 1991), which is applied in European
Community since 1 January 2002 (the European 91/180.CEE, EC Regulation Nr. 1664/2006).
Two tests were successively used: Acidification test based on the identification of a possible
inhibition of B. stearothermophillus variety calidolactis ATCC 10149, as indicated by the
turn indicator colored antibiotic residues that would be present in the sample, followed by a
confirmation test, corresponding to the realization of three agar diffusion tests using tree
bacteria strains: B. stearothermophilus, B. subtilis and B. megaterium.
Acidification test: This test allows an initial screening of all the samples tested. It is based
on the addition in the milk sample of nutrient mixture with agar containing a pH indicator and
spores of B. stearothermophillus (variety calidolactis, ATCC 10149, purchased from Pasteur
Intitute from Algeria). Generally, the strain present a very good sensitivity and more
particularly for penicillin. Normal growth and acid production by this organism after incubation
causes a color change of the pH indicator (bromocresol purple) (SIGMA ALDRICH, Germany)
which turns from purple to yellow. When milk contains inhibitory substances, the growth of the
test organism is affected; the color of the pH indicator remains purple. A negative control (milk
free from antibiotics) and a positive control (milk containing penicillin) are subjected to the test
under the same conditions and at the same time as the tested samples.
Agar diffusion test: all positive milk samples or doubtful obtained by acidification
test will be checked by confirmatory test. This test consists of using three agar diffusion
tests inoculated with spores of B. stearothermophillus, B. subtilis and B. megaterium. The
Muller-Hinton agar was previously melted at 100C and cooled at 55 C before being
poured into Petri dishes. The medium was inoculated with 103 to 105 spores B.
stearothermophillus, B. subtilis and B. megaterium/ml. After preparing a sterile paper
disc, the latest were impregnated with milk samples and deposited on the agar surface.
The Petri dishes were incubated at 55C for B. stearothermophillus and 37C for B.
subtilis and B. megaterium. After 24h of incubation, the diameters of inhibition areas were
measured by using calipers. The different families of antibiotics detected by inhibition of
this microorganisms test are summarized in table 1.

RESULTS AND DISCUSSIONS

The results of this study showed after using acidification test (first stage), the
presence of antibiotics residues in 115 of the total 171 raw milk samples examined

84
(67.25%), reflecting their high presence in the milk collection. In contrast, the change of
middle coloration from purple to yellow was only observed in 49 samples (28.65%)
which were considered negative. It was impossible to confirm their presence in 7 milk
samples (4.09%) (Table 2). The last samples were considered doubtful. That is the reason
we needed a confirmation test.
Table 1
Detection of several antibiotic residues in raw milk samples
Microorganism Families antibiotics Antibiotic
Test
Bacillus stearothermophillus Penicillins Penicillin G
Tetracyclines Tetracycline
Bacillus subtilis Macrolides Spiramycin
Aminosides Erythromycin
Streptomicin
Bacillus megaterium Sulfamides Trimethoprim sulfamethoxazole

Table 2
Results of analyzing raw milk samples after using acidification test
Number of RESULTS
all samples Number of Number of negatives Number of doubtful
examined positives cases cases cases
171 115 (67.25%) 49 (28.65%) 7 (4.09%)

In addition, the confirmation test showed that presence of antibiotics residues

recorded only 80 against 91 raw milk samples examined, 46.78% vs 53.21% respectively
(Table 3). The samples were considerate free from antibiotic residues.
Table 3
Results of analysis of raw milk after using confirmatory test
Number of samples Results
Examined Number and percentage Number and percentage
of positive cases of negative cases
171 80 (46.78%) 91 (53.21%)

Table 4
Percentage per families of antibiotics residues in raw milk samples
Number of positive Result:
raw milk samples examined (Number and percentage of positive cases)
Penicillins and/or Macrolides and/or Sulfonamides
80 Tetracyclines Aminoglycosides
71 (88.75%) 10 (12.5%) 4 (5%)

However, most positive raw milk samples showed a high percentage of contamination
with penicillin and/or tetracycline (88.75%). Conversely, the macrolides and/or
aminoglycosides were detected in only 10 over 80 positive samples (12.5%) and for
Sulfonamides in only 5% of positive cases (4 over 80 positive samples) (Table 4). We noticed
that only 10 over 71 samples contaminated by penicillin and/or tetracycline presented greater
diameter of inhibition (more than 151mm) for B. stearothermophillus. The first screening
test (acidification) revealed that 115 over 171 raw milk samples examined tested positive for

85
antibiotics residues. However this test was not able to conclude definitively the results of
some samples qualified as doubtful, hence the need for a confirmation test. The agar diffusion
technique, using the three strains, B. stearo thermophillus, B.subtilis and B. megaterium, has
lifted any ambiguity for these doubtful samples. The choice of B. stearothermophillus as test
strain is definitely better than the use of other strains such as S. aureus, which showed a lot of
limiting factors such as in terms of susceptibility to antibiotics (low zone of inhibition at high
concentrations of antibiotics), the risk of resistance (secretion of penicillinase by the
microorganism or by mutation) and inhibition by fermenters germs (Lactobacillus lactis,
Streptococcus lactis, Streptococcus thermophillus) (Hilan and Chemali., 1998). Indeed, B.
stearothermophillus is characterized by a remarkable sensitivity to lactam antibiotic; this
growth is inhibited by 5 ppb of concentration of ampicillin. The sensitivity of the acidification
test is particularly high for cloxacillin and reasonably acceptable for tetracycline.
The acidification test is thus characterized by sensitivity close to MRL for cloxacillin
and 50-100% of MRLs for tetracycline compared to the old method using Streptococcus
thermophillus as test microorganism, that offers to both antibiotics mentioned above that
sensitivities of 3 to 4 times the MRL and 2-4 times MRL (Navratilova, 2008).
The risk to have false negative results with milk at concentrations of antibiotic
residues near very low for MRLs is thus limited, thus the confirmatory test shows a high
specificity. Our results were not in agreement with those reported by Ben Mahdi and
Ouslimani (2009). In fact, their results indicate a low level of contamination rate (9.87%).
This difference could be explained by the relatively small number of our samples. The results
were similar to those reported by Zinedine et al. (2007) who obtained 57.14% positive cases.
However, the percentage of presence of antibiotic residues in raw milk varied in some studies.
In fact, Srairi et al (2004), Kivaria et al (2006), Adesiyun et al (2007) and Kouame et al
(2010) have obtained 25, 7, 6.5 and respectively 24.7% positive cases.
The high percentage of contamination of the milk by inhibitors such as antibiotic
residues, can be probably explained mainly by massive and uncontrolled intra-mammary
pharmaceutical preparations for the treatment and prevention of bovine mastitis, not
respecting the waiting times after treatment and secondly by a voluntary addition of germs
growth inhibitors (antibiotics, antiseptics) in order to stop microbial growth and stabilize
the microbial quality of milk (Zinedine et al., 2007). The high contamination of milk
samples in tetracycline and/or penicillin was also confirmed by Ben Mahdi and Ouslimani
(2009) who reported their presence in 97.33% raw milk samples. According to Ameur et
al. (2008), in our area and other part of Tizi-Ouzou area like Azazga and Yakouren, the
use of intra- mammary syringes is often aiming to prevent acute mastitis. The most
prescribed and used products contain tetracycline, penicillin and rarely mcrolides. The
choice of these molecules is mainly based on their efficiency and their low price.
In addition to the risk to public health (allergic reactions, influence on the intestinal
flora and the risk of emergence and increase of antibiotic-resistant strains), the antibiotic
residues represent a real problem for milk processors because of their negative impact on the
process of lactic fermentation. In fact, lactic acid bacteria like Streptococcus thermophillus,
Lactobacillus bulgaricus and Lactococcus lactis can play an essential role as starter by
acidifying the milk, there by resulting in the precipitation of milk protein (casein), the
development of aromas linked to proteolytic activity and lipolytic lactic strains and the
inhibition of the growth of alteration microorganisms or potentially pathogenic bacteria such
as coliforms, Pseudomonas, S. aureus and Listeria monocytogenes (Chamba, 2008). The
presence of antibiotic residues in the raw milk may partially or completely inhibit the growth
of lactic acid bacteria involved in the development of dairy products such as cheese and
yogurt, leading to manufacture accidents. The most common accidents are the milk

86
 coagulation defects, the inadequate draining and the risk of uncontrolled proliferation of
gasifiers germs, insensitive to antibiotics, such as coliforms, Bacillus, Clostridium and
Proteus (Berger and Lenoir, 1997). This problem generates economic losses every year.

CONCLUSION

The results of this study indicate clearly the high contamination of our raw milk
samples by antibiotic residues. This can be explained by the intense and misuse of
veterinary drugs, as well as the non respect of time between the administration of the
antibiotic to patient and the milk collection. To solve this situation, certain measures must
be applied, such as: the respect of good veterinary practices, the respect of withdrawal time
before the collection of milk product, and a regular and systematic screning of antibiotic
residues in milk before consumption or technological use. Finally, we hope to lead other
studies in the identification of antibiotic residues in milk, using a combination of two
techniques: the microbiological method with agar diffusion and the analytical method (high
performance liquid chromatography) to determine not only the type of antibiotic residues
used but also their concentration in the milk collected.

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